The activation mechanism of ACK1 (activated Cdc42-associated tyrosine kinase 1)

ACK [activated Cdc42 (cell division cycle 42)-associated tyrosine kinase; also called TNK2 (tyrosine kinase, non-receptor, 2)] is activated in response to multiple cellular signals, including cell adhesion, growth factor receptors and heterotrimeric G-protein-coupled receptor signalling. However, the molecular mechanism underlying activation of ACK remains largely unclear. In the present study, we demonstrated that interaction of the SH3 (Src homology 3) domain with the EBD [EGFR (epidermal growth factor receptor)-binding domain] in ACK1 forms an auto-inhibition of the kinase activity. Release of this auto-inhibition is a key step for activation of ACK1. Mutation of the SH3 domain caused activation of ACK1, independent of cell adhesion, suggesting that cell adhesion-mediated activation of ACK1 is through releasing the auto-inhibition. A region at the N-terminus of ACK1 (Leu10-Leu14) is essential for cell adhesion-mediated activation. In the activation of ACK1 by EGFR signalling, Grb2 (growth-factor-receptor-bound protein 2) mediates the interaction of ACK1 with EGFR through binding to the EBD and activates ACK1 by releasing the auto-inhibition. Furthermore, we found that mutation of Ser445 to proline caused constitutive activation of ACK1. Taken together, our studies have revealed a novel molecular mechanism underlying activation of ACK1.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5'-(beta,gamma-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623-652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284 in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.     

Identification of the region in Cdc42 that confers the binding specificity to activated Cdc42-associated kinase

The Rho family small G-protein Cdc42 has been implicated in a diversity of biological functions. Multiple downstream effectors have been identified. How Cdc42 discriminates the interaction with its multiple downstream effectors is not known. Activated Cdc42-associated tyrosine kinase (ACK) is a very specific effector of Cdc42. To delineate the Cdc42 signaling pathway mediated by ACK, we set about to identify the specific ACK-binding region in Cdc42. We utilized TC10, another member of the Rho family of G-proteins that is 66.7% identical to Cdc42, to construct TC10/Cdc42 chimeras for screening the specific ACK-binding region in Cdc42. A region between switch I and switch II has been identified as the specific ACK-binding (AB) region. The replacement of the AB region with the corresponding region in TC10 resulted in the complete loss of ACK-binding ability but did not affect the binding to WASP, suggesting that the AB region confers the binding specificity to ACK. On the other hand, replacement of the corresponding region of TC10 with the AB region enabled TC10 to acquire ACK-binding ability. Eight residues are different between the AB region and the corresponding region of TC10. The mutational analysis indicated that all eight residues contribute to the binding to ACK2. The assays for the Cdc42-mediated activation of ACK2 indicated that the AB region is essential for Cdc42 to activate ACK2 in cells. Thus, our studies have defined a specific ACK-binding region in Cdc42 and have provided a molecular basis for generating ACK binding-defective mutants of Cdc42 to delineate ACK-mediated signaling pathway.     

Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

The nonreceptor tyrosine kinase ACK2, a specific target for Cdc42 and a negative regulator of cell growth and focal adhesion complexes

ACK2 (activated Cdc42-associated tyrosine kinase-2) is a nonreceptor tyrosine kinase that is a specific target/effector for the GTP-binding protein Cdc42. Thus far the biological function of this tyrosine kinase has not been determined. Using an inducible eukaryotic expression system in fibroblasts, we demonstrate that ACK2 can strongly influence cell shape and growth as well as focal complex formation. ACK2 was found to associate with the focal adhesion complex components talin and vinculin, but not with the focal adhesion kinase (FAK), in a kinase-independent manner. The tyrosine kinase activity of FAK was also inhibited in cells overexpressing both wild-type and kinase-defective ACK2. This may be due to a competition between ACK2 and FAK for Src, which is an essential cofactor for FAK activation, as we have found that ACK2 specifically binds Src in cells. The ACK2-Src interaction appears to be mediated by the SH3 domain of Src, and the phosphorylation of ACK2 is enhanced in cells overexpressing the hyperactivated Src(Y527F) mutant. Overexpression of both wild-type and kinase-defective ACK2 also results in a severe inhibition of cell growth. In addition, ACK2 dissolves actin stress fibers and disassembles focal complexes but in a kinase-dependent manner. These results, taken together with previous studies demonstrating an association of ACK2 with integrin beta(1) (Yang, W., Lin, Q., Guan, J.-L., Cerione, R. A. (1999) J. Biol. Chem. 274, 8524-8530) and clathrin (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473), suggest that the binding and protein tyrosine kinase activities of ACK2 coordinate changes in cell morphology and growth with the disassembly of focal adhesion sites, perhaps to organize new integrin complexes that are required for endocytosis and/or for cellular differentiation.     

Activation of the Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion via integrin beta1

Activated Cdc42-associated kinase-2 (ACK-2) is a non-receptor tyrosine kinase that appears to be a highly specific target for the Rho-related GTP-binding protein Cdc42. In order to understand better how ACK-2 activity is regulated in cells, we have expressed epitope-tagged forms of this tyrosine kinase in COS-7 and NIH3T3 cells. We find that ACK-2 can be activated by cell adhesion in a Cdc42-dependent manner. However, unlike the focal adhesion kinase, which also is activated by cell adhesion, the activation of ACK-2 is F-actin-independent and does not require cell spreading. In addition, overexpression of ACK-2 in COS-7 cells did not result in the stimulation of extracellular signal-regulated kinase activity but rather activated the c-Jun kinase. Both anti-integrin beta1 antibody and RGD peptides inhibited the activation of ACK-2 by cell adhesion. In addition, ACK-2 was co-immunoprecipitated with integrin beta1. Overall, these findings suggest that ACK-2 interacts with integrin complexes and mediates cell adhesion signals in a Cdc42-dependent manner.     

Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase

Treatment of cells with epidermal growth factor (EGF) promotes the activation of the small GTP-binding protein Cdc42, as well as its phosphorylation in cells. The EGF-dependent phosphorylation of Cdc42 occurs at tyrosine 64 in the Switch II domain and appears to be mediated through the Src tyrosine kinase, because both the expression of a dominant-negative Src mutant (mouse Src(K297R)) and treatment of cells with the Src kinase inhibitor PP2 blocks the EGF-stimulated phosphorylation of Cdc42, whereas expression of an activated Src mutant (Src(Y529F)) promotes phosphorylation in the absence of EGF treatment. The EGF-stimulated phosphorylation of Cdc42 is not required for its activation, nor does it directly affect the interactions of activated Cdc42 with target/effector proteins including PAK, ACK, WASP, or IQGAP. However, the EGF-stimulated phosphorylation of Cdc42 is accompanied by an enhancement in the interaction of Cdc42 with the Rho-GDP dissociation inhibitor (RhoGDI). The EGF-stimulated activation of Cdc42 does require activated Src, as well as the Vav2 protein, a member of the Dbl family of guanine nucleotide exchange factors. Src catalyzes the tyrosine phosphorylation of Vav2, and overexpression of Vav2 together with activated Src (Src(Y529F)) can completely bypass the need for EGF to promote the activation of Cdc42. Thus, EGF signaling through Src appears to have dual regulatory effects on Cdc42: 1). it leads to the activation of Cdc42 as mediated by the Vav2 guanine nucleotide exchange factor, and 2). it results in the phosphorylation of Cdc42, which stimulates the binding of RhoGDI, perhaps to direct the movement of Cdc42 to a specific cellular site to trigger a signaling response, because Cdc42-RhoGDI interactions are essential for Cdc42-induced cellular transformation.     

Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast

Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.     

Requirement of activated Cdc42-associated kinase for survival of v-Ras-transformed mammalian cells

Activated Cdc42-associated kinase (ACK) has been shown to be an important effector molecule for the small GTPase Cdc42. We have shown previously an essential role for Cdc42 in the transduction of Ras signals for the transformation of mammalian cells. In this report, we show that the ACK-1 isoform of ACK plays a critical role in transducing Ras-Cdc42 signals in the NIH 3T3 cells. Overexpression of a dominant-negative (K214R) mutant of ACK-1 inhibits Ras-induced up-regulation of c-fos and inhibits the growth of v-Ras-transformed NIH 3T3 cells. Using small interfering RNA, we knocked down the expression of ACK-1 in both v-Ha-Ras-transformed and parental NIH 3T3 cells and found that down-regulation of ACK-1 inhibited cell growth by inducing apoptosis only in v-Ha-Ras-transformed but not parental NIH 3T3 cells. In addition, we studied the effect of several tyrosine kinase inhibitors and found that PD158780 inhibits the kinase activity of ACK-1 in vitro. We also found that PD158780 inhibits the growth of v-Ha-Ras-transformed NIH 3T3 cells. Taken together, our results suggest that ACK-1 kinase plays an important role in the survival of v-Ha-Ras-transformed cells, suggesting that ACK-1 is a novel target for therapies directed at Ras-induced cancer.     

Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway

The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho family GTPase Cdc42. GTP-bound Cdc42 has been shown to facilitate neurite outgrowth elicited by activation of muscarinic cholinergic receptors (mAChRs). Because tyrosine kinase activity is a requirement for neuritogenesis in several cell systems, we investigated whether endogenous mAChRs (principally of the M3 subtype) expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1. Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold increase in the tyrosine phosphorylation of ACK-1 which was inhibited by atropine. ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, an inhibitor of Rho family GTPases. In contrast, disruption of the actin cytoskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreover, addition of Oxo-M to cells preincubated with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1. Although stimulation of M3 mAChRs induces both an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation. Instead, inhibition of PKC resulted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC with phorbol ester decreased ACK-1 phosphorylation. The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in an agonist-dependent manner. Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphorylation. The data are the first to show phosphorylation of ACK-1 after stimulation of a receptor coupled to neurite outgrowth and indicate that a Rho family GTPase (i.e. Cdc42) and Fyn are essential upstream elements of this signaling pathway.     

SNX9 as an adaptor for linking synaptojanin-1 to the Cdc42 effector ACK1

Sorting nexin 9 (SNX9, also referred to as SH3PX1) is a binding partner for the non-receptor and Cdc42-associated kinase (ACK) in Drosophila and mammals. ACK1 is known to bind clathrin and influence EGF receptor endocytosis. SNX9 comprises an N-terminal Src homology domain 3 (SH3), a central PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. In order to investigate SNX9 further we have made use of a novel in vivo biotinylation system to label various GST-SH3 domains and perform blot overlays, thereby identifying synaptojanin-1 as a partner for SNX9. Biotinylated SH3 domains were also used for specific identification of target proline-rich sequences in synaptojanin and ACK1 on synthetic peptides arrays. Direct assessment of SH3 binding efficiencies at different positions within the extensive proline-rich regions of these proteins were thus determined. While SNX9 targets a number of sequences within the proline-rich regions of synaptojanin, a single site was identified in human ACK1. By testing the association of various truncations of ACK1 with SNX9 we confirmed the dominant SNX9 binding domain in human ACK1 (residues 920-955). In the presence of SNX9 we find that synaptojanin is able to colocalize with distinct ACK1 containing vesicles, indicating that this tyrosine kinase is linked to many components involved in vesicle dynamics including clathrin, AP2 and synaptojanin-1.     

Expression pattern of ACK1 tyrosine kinase during brain development in the mouse

Ack1 is a non-receptor tyrosine kinase that is highly expressed in the adult central nervous system (CNS). Here, we studied the distribution of Ack1 mRNA throughout the development of mouse CNS. Expression was detected in all areas of the brain but especially high levels were observed in the neocortex, hippocampus, and cerebellum. Interestingly, expression levels were prominent in areas of proliferation such as the subventricular zone and areas that originate other structures such the pontine nucleus and the ganglionic eminence. During development, several areas showed an increase in Ack1 expression, especially the dentate gyrus and CA3 in the hippocampus, layer V in the neocortex, and the Purkinje cell layer in the cerebellum. These results demonstrate that this kinase is up-regulated during development and that it is expressed in proliferative areas and in migratory pathways in the developing brain.     

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.     

Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner

The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins. However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood. Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements. The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively. Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF. Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell. In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed. The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho. These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl.     

Phosphorylation of Bruton's tyrosine kinase by c-Abl

Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.